Comparable atherosclerotic plaque areas in the aortas of <i>WT→ Ldlr</i>^{<i>–/–</i>}and <i>Dgat1</i>^{<i>–/–</i>}<i>→ Ldlr</i>^{<i>–/–</i>}mice.

<p>(A) Aortae stained <i>en face</i> with ORO after 13 and 19 weeks of WTD feeding. (B) Quantification of plaque size in the thoracic aortae and (C) aortic arches of <i>WT→ Ldlr</i>^{<i>–/–</i>}(black squares) and <i>Dgat1</i>^{<i>–/–</i>}<i>→ Ldlr</i>^{<i>–/–</i>}(white circles) mice fed a WTD as in A (n = 9 females, ♀, and 10–11 males, ♂). Horizontal lines represent mean values through data groupings.</p

Increased plaque inflammation in <i>Dgat1</i>^{<i>–/–</i>}<i>→ Ldlr</i>^{<i>–/–</i>}mice.

<p>(A-C, left) Representative images of aortic root sections stained with ORO, MoMa-2, and Masson's Trichrome. (A-C, right) Quantification of these images to measure plaque size, macrophage content, and collagen deposition, respectively. (D-F) Integrated quantification of data from A-C to determine collagen-to-macrophage ratios, necrotic core per plaque area, and collagen-to-necrotic core ratios, respectively. (G) Representative images of Masson’s Trichrome-stained aortic root sections focused on plaque fibrotic caps, with quantification of minimal fibrotic cap thickness. All data represent means ± SEM of 12 aortic root sections for ORO and three aortic root sections in the area of maximal plaque size for MoMa-2- and Trichrome-staining per mouse after 13 (n = 9 females, ♀) and 19 weeks (n = 10–11 males, ♂) of WTD feeding. *, p < 0.05.</p

Altered circulating immune cell numbers in <i>Dgat1</i>^{<i>–/–</i>}<i>→ Ldlr</i>^{<i>–/–</i>}mice fed a WTD.

<p>(A) Total leukocyte counts, and relative (B) monocyte, (C) eosinophil, (D) basophil, (E) lymphocyte, and (F) neutrophil counts in both <i>WT→ Ldlr</i>^{<i>–/–</i>}and <i>Dgat1</i>^{<i>–/–</i>}<i>→ Ldlr</i>^{<i>–/–</i>}after 13 weeks (females, ♀) and 19 weeks (males, ♂) of WTD feeding (n = 9 per group), showing reduced lymphocyte and increased neutrophil counts in male mice, with a similar trend in female mice. Data are presented as mean ± SEM. **, p ≤ 0.01; ***, p ≤ 0.001.</p

Plasma parameters.

<p>Plasma parameters.</p

<i>Dgat1</i>^{<i>–/–</i>}<i>→ Ldlr</i>^{<i>–/–</i>}and <i>WT→ Ldlr</i>^{<i>–/–</i>}mice have comparable body weight gain and adiposity.

<p>Weight gain of (A) female mice (n = 9) and (B) male mice (n = 10–11) fed a WTD. (C) Total body fat, (D) relative adiposity, (E) lean mass, (F) gonadal fat pad weights, and (G) liver weights of <i>WT→ Ldlr</i>^<i>-/-</i> and <i>Dgat1</i>^<i>-/-</i><i>→ Ldlr</i>^<i>-/-</i> mice after 13 (n = 9 female mice, ♀) and 19 weeks (n = 10–11 male mice, ♂) of WTD, respectively. For all panels, data are presented as means ± SEM.</p

M₁ and M₂ gene expression in lipoprotein-treated <i>WT</i> and <i>Dgat1</i>^{<i>–/–</i>}macrophages.

<p>qPCR analysis of control macrophages vs. those treated with 100 μg/ml VLDL, 100 μg/ml acLDL or 100 ng/ml LPS (positive control for M₁ activation). Shown are mRNA levels of (A) M₁ marker and (B) M₂ marker genes analyzed in duplicate. Data are means (n = 3–6) ± SEM. *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.</p

Transition between Acute and Chronic Hepatotoxicity in Mice Is Associated with Impaired Energy Metabolism and Induction of Mitochondrial Heme Oxygenase-1

<div><p>The formation of protein inclusions is frequently associated with chronic metabolic diseases. In mice, short-term intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to hepatocellular damage indicated by elevated serum liver enzyme activities, whereas only minor morphological changes are observed. Conversely, chronic administration of DDC for several weeks results in severe morphological damage, characterized by hepatocellular ballooning, disruption of the intermediate filament cytoskeleton, and formation of Mallory-Denk bodies consisting predominantly of misfolded keratins, Sqstm1/p62, and heat shock proteins. To evaluate the mechanistic underpinnings for this dichotomy we dissected the time-course of DDC intoxication for up to 10 weeks. We determined body weight change, serum liver enzyme activities, morphologic alterations, induction of antioxidant response (heme oxygenase-1, HO-1), oxidative damage and ATP content in livers as well as respiration, oxidative damage and the presence and activity of HO-1 in endoplasmic reticulum and mitochondria (mtHO-1). Elevated serum liver enzyme activity and oxidative liver damage were already present at early intoxication stages without further subsequent increase. After 2 weeks of intoxication, mice had transiently lost 9% of their body weight, liver ATP-content was reduced to 58% of controls, succinate-driven respiration was uncoupled from ATP-production and antioxidant response was associated with the appearance of catalytically active mtHO-1. Oxidative damage was associated with both acute and chronic DDC toxicity whereas the onset of chronic intoxication was specifically associated with mitochondrial dysfunction which was maximal after 2 weeks of intoxication. At this transition stage, adaptive responses involving mtHO-1 were induced, indirectly leading to improved respiration and preventing further drop of ATP levels. Our observations clearly demonstrate principally different mechanisms for acute and chronic toxic damage.</p></div

Change of body weight, hepatic ATP content and mitochondrial respiration and respiration control ratio (RCR) by DDC-intoxication.

<p>A: Percent body weight change, compared to week 0, monitored for each mouse over 10 weeks in the control and DDC intoxication groups. Data are mean ± SD of 5 mice per treatment group. B: Hepatic ATP content was quantified in control and DDC-treated mice. Each data point is the mean ± SD from three mice per treatment group. C: Respiration control ratio (RCR) with 5 mM succinate as substrate. D: RCR using 2.5 mM each glutamate/malate as substrate. E: Respiration rates with 5 mM succinate in absence (state4) and presence (state3) of 250 µM ADP. F: Respiration rates with 2.5 mM each glutamate/malate in absence (state4) and presence (state3) of 250 µM ADP. Data are mean ± SD from 6 mice per treatment group. Significances are shown versus controls: * p<0.05; ** p<0.01.</p

Expression of Nrf2 and heme oxygenase 1 in livers of DDC-treated mice.

<p>A: Nrf2 protein expression and (B) densitometry in hepatic nuclear fractions. The intensity of protein bands was quantified, and individual blot densities were normalized to loading control (Lamin A/C) C: Heme oxygenase-1 (HO-1) protein expression and (D) densitometry in total liver homogenate (HO-1) and mitochondrial fractions (mtHO-1). The intensity of protein bands was quantified, and individual blot densities were normalized to loading control (calnexin for total homogenate, Ponceau S for the mitochondrial fraction). (E) Activities of HO-1 in microsomes and mtHO-1 in mitochondria as pmol bilirubin/mg protein/h.</p

Oxidative damage to liver tissue during DDC-intoxication.

<p>MDA-protein adduct levels (pmol/mg protein) in (A) liver homogenate and (B) mitochondria. Concentrations of 8-OHdG (ng/µg DNA) in (C) liver homogenate, representing total DNA, and (D) mitochondria. Data are mean ± SD from 5 mice per treatment group. Significant differences were found compared to controls (*** p<0.001), but not between treatment groups.</p